Modulation of exon recognition in pre-mrna by interfering with the secondary rna structure

ABSTRACT

The invention relates to oligonucleotides for inducing skipping of exon 53 of the dystrophin gene. The invention also relates to methods of inducing exon 53 skipping using the oligonucleotides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of patent application Ser. No.14/688,871 filed Apr. 16, 2015, which is a continuation of patentapplication Ser. No. 14/295,298 filed Jun. 3, 2014 and a continuation ofpatent application Ser. No. 13/718,666 filed Dec. 18, 2012, the latterof which is a continuation of patent application Ser. No. 13/550,210,filed Jul. 16, 2012, which is a continuation of U.S. Pat. No. 8,759,507(formerly U.S. patent application Ser. No. 12/976,381, filed Dec. 22,2010, which is a continuation of U.S. Pat. No. 7,534,879 (formerly U.S.patent application Ser. No. 12/198,007, filed Aug. 25, 2008), which is acontinuation of patent application Ser. No. 11/233,507, filed on Sep.21, 2005, and a continuation of patent application Ser. No. 11/233,495,filed on Sep. 21, 2005, the latter of which claims the benefit ofInternational Patent Application No. PCT/NL2003/000214, filed Mar. 21,2003, the contents of all of which are hereby incorporated by referencein their entirety.

SEQUENCE LISTING

The attached sequence listing is hereby incorporated by reference.

TECHNICAL FIELD

The invention relates to the fields of molecular biology and medicine.More in particular, the invention relates to the restructuring of mRNAproduced from pre-mRNA, and therapeutic uses thereof.

BACKGROUND

The central dogma of biology is that genetic information resides in theDNA of a cell and is expressed upon transcription of this information,where after production of the encoded protein follows by the translationmachinery of the cell. This view of the flow of genetic information hasprompted the predominantly DNA-based approach for ‘interfering with theprotein content of a cell. This view is slowly changing and alternativesfor interfering at the DNA level are being pursued.

In higher eukaryotes, the genetic information for proteins in the DNA ofthe cell is encoded in exons that are separated from each other byintronic sequences. These introns are in some cases very long. Thetranscription machinery generates a pre-mRNA that contains both exonsand introns, while the splicing machinery, often already during theproduction of the pre-mRNA, generates the actual coding region for theprotein by splicing together the exons present in the pre-mRNA,

Although much is known about the actual processes involved in thegeneration of an mRNA from a pre-mRNA, much also remains hidden. In theinvention it has been shown possible to influence the splicing processsuch that a different mRNA is produced. The process allows for thepredictable and reproducible restructuring of mRNA produced by asplicing machinery. An oligonucleotide capable of hybridizing topre-mRNA at a location of an exon that is normally included in themature mRNA can direct the exclusion of the thus targeted exon or a partthereof.

SUMMARY OF THE INVENTION

In the invention, means and methods are provided for the design ofappropriate complementary oligonucleotides. To this end, the inventionprovides a method for generating an oligonucleotide comprisingdetermining, from a (predicted) secondary structure of RNA from an exon,a region that assumes a structure that is hybridized to another part ofthe RNA (closed structure) and a region that is not hybridized in thestructure (open structure), and subsequently generating anoligonucleotide, which at least in part is complementary to the closedstructure and which at least in part is complementary to the openstructure. RNA molecules exhibit strong secondary structures, mostly dueto base pairing of complementary or partly complementary stretcheswithin the same RNA. It has long since been thought that structures inthe RNA play a role in the function of the RNA. Without being bound bytheory, it is believed that the secondary structure of the RNA of anexon plays a role in structuring the splicing process. Through itsstructure, an exon is recognized as a part that needs to be included inthe pre-mRNA. Herein, this signaling function is referred to as an “exoninclusion signal.” A complementary oligonucleotide of the invention iscapable of interfering with the structure of the exon and therebycapable of interfering with the exon inclusion signal of the exon. Ithas been found that many complementary oligonucleotides indeed comprisethis capacity, some more efficiently than others. Oligonucleotides ofthe invention, i.e., those with the overlap directed toward open andclosed structures in the native exon RNA, are a selection from allpossible oligonucleotides. The selection encompasses oligonucleotidesthat can efficiently interfere with an exon inclusion signal.

Without being bound by theory, it is thought that the overlap with anopen structure improves the invasion efficiency of the oligonucleotide(i.e, increases the efficiency with which the oligonucleotide can enterthe structure), whereas the overlap with the closed structuresubsequently increases the efficiency of interfering with the secondarystructure of the RNA of the exon, and thereby interfere with the exoninclusion signal. It is found that the length of the partialcomplementarity to both the closed and the open structure is notextremely restricted. We have observed high efficiencies witholigonucleotides with variable lengths of complementarily in eitherstructure. The term “complementarity” is used herein to refer to astretch of nucleic acids that can hybridize to another stretch ofnucleic acids under physiological conditions. It is thus not absolutelyrequired that all the bases in the region of complementarity are capableof pairing with bases in the opposing strand. For instance, whendesigning the oligonucleotide, one may want to incorporate, for example,a residue that does not base pair with the base on the complementarystrand. Mismatches may to some extent be allowed, if under thecircumstances in the cell, the stretch of nucleotides is capable ofhybridizing to the complementary part. In a preferred embodiment, acomplementary part (either to the open or to the closed structure)comprises at least three and more preferably, at least four, consecutivenucleotides. The complementary regions are preferably designed such thatwhen combined, they are specific for the exon in the pre-mRNA. Suchspecificity may be created with various lengths of complementary regionsas this depends on the actual sequences in other mRNA or pre-mRNA in thesystem. The risk that also one or more other pre-mRNA will be able tohybridize to the oligonucleotide decreases with increasing size of theoligonucleotide. It is clear that oligonucleotides comprising mismatchesin the region of complementarity but that retain the capacity tohybridize to the targeted region(s) in the pre-mRNA, can be used in theinvention, However, preferably at least the complementary parts do notcomprise such mismatches as these typically have a higher efficiency anda higher specificity, than oligonucleotides having such mismatches inone or more complementary regions. It is thought that higherhybridization strengths (i.e., increasing number of interactions withthe opposing strand), are favorable in increasing the efficiency of theprocess of interfering with the splicing machinery of the system.

The secondary structure is best analyzed in the context of the pre-mRNAwherein the exon resides. Such structure may be analyzed in the actualRNA. However, it is currently possible to predict the secondarystructure of an RNA molecule (at lowest energy costs) quite well usingstructure-modeling programs. A non-limiting example of a suitableprogram is RNA mfold version 3.1 server (Mathews et al., 1999, J. Mol.Biol. 288:911-940). A person skilled in the art will be able to predict,with suitable reproducibility, a likely structure of the exon, given thenucleotide sequence. Best predictions are obtained when providing suchmodeling programs with both the exon and flanking intron sequences. Itis typically not necessary to model the structure of the entirepre-mRNA.

The open and closed structure to which the oligonucleotide is directed,are preferably adjacent to one another. It is thought that in this way,the annealing of the oligonucleotide to the open structure inducesopening of the closed structure, and annealing progresses into thisclosed structure. Through this action, the previously closed structureassumes a different conformation. The different conformation may resultin the disruption of the exon inclusion signal. However, when potential(cryptic) splice acceptor and/or donor sequences are present within thetargeted exon, occasionally a new exon inclusion signal is generateddefining a different (neo) exon, e.g., with a different 5′ end, adifferent 3′ end, or both. This type of activity is within the scope ofthe invention as the targeted exon is excluded from the mRNA. Thepresence of a new exon, containing part of the targeted exon, in themRNA does not alter the fact that the targeted exon, as such, isexcluded. The inclusion of a neo-exon can be seen as a side effect whichoccurs only occasionally. There are two possibilities when exon skippingis used to restore (part of) an open reading frame that was disrupted asa result of a mutation. One is that the neo-exon is functional in therestoration of the reading frame, whereas in the other case the readingframe is not restored. When selecting oligonucleotides for restoringreading frames by means of exon-skipping, it is, of course, clear thatunder these conditions, only those oligonucleotides are selected thatindeed result in exon-skipping that restores the open reading frame,with or without a neo-exon.

Pre-mRNA can be subject to various splicing events, for instance,through alternative splicing. Such events may be induced or catalyzed bythe environment of a cell or artificial splicing system. Thus, from thesame pre-mRNA, several different mRNAs may be produced. The differentmRNAs all included exonic sequences, as that is the definition of anexon. However, the fluidity of the mRNA content necessitates adefinition of the term “exon” in the invention. An “exon,” according tothe invention, is a sequence present in both the pre-mRNA and mRNAproduced thereof, wherein the sequence included in the mRNA is, in thepre-rriRNA, flanked on one side (first and last exon) or both sides (anyexon other than the first and the last exon) by sequences not present inthe mRNA. In principle, any rnRNA produced from the pre-mRNA qualifiesfor this definition. However, for the invention, so-called dominantmRNAs are preferred, i.e., mRNA that makes up at least 5% of the mRNAproduced from the pre-mRNA under the set conditions. Humanimmunodeficiency virus, in particular, uses alternative splicing to anextreme. Some very important protein products are produced from mRNAmaking up even less than 5% of the total mRNA produced from the virus.The genomic RNA of retroviruses can be seen as pre-mRNA for any splicedproduct derived from it. As alternative splicing may vary in differentcell types, the exons are defined as exons in the context of thesplicing conditions used in that system. As a hypothetical example, anmRNA in a muscle cell may contain an exon that is absent in an mRNAproduced from the same pre-mRNA in a nerve cell. Similarly, mRNA in acancer cell may contain an exon not present in mRNA produced from thesame mRNA in a normal cell.

Alternative splicing may occur by splicing from the same pre-mRNA.However, alternative splicing may also occur through a mutation in thepre-mRNA, for instance, generating an additional splice acceptor and/orsplice donor sequence. Such alternative splice sequences are oftenreferred to as cryptic splice acceptor/donor sequences. Such crypticsplice sites can result in new exons (neo-exons). Inclusion of neo-exonsinto produced mRNA can be prevented, at least in part, using a method ofthe invention. In case a neo-exon is flanked by a cryptic and a “normal”splice donor/acceptor sequence, the neo-exon encompasses the old (paleo)exon. If in this case the original splice donor/acceptor sequence, forwhich the cryptic splice donor/acceptor has taken its place, is stillpresent in the pre-mRNA, it is possible to enhance the production ofmRNA containing the paleo-exon by interfering with the exon-recognitionsignal of the neo-exon. This interference can be both in the part of theneo-exon corresponding to the paleo-exon, or the additional part of suchneo-exons. This type of exon skipping can be seen as splice correction.

The exon skipping technique can be used for many different purposes.Preferably, however, exon skipping is used for restructuring mRNA thatis produced from pre-mRNA exhibiting undesired splicing in a subject.The restructuring may be used to decrease the amount of protein producedby the cell. This is useful when the cell produces a particularundesired protein. In a preferred embodiment, however, restructuring isused to promote the production of a functional protein in a cell, i.e.,restructuring leads to the generation of a coding region for afunctional protein. The latter embodiment is preferably used to restorean open reading frame that was lost as a result of a mutation. Preferredgenes comprise a Duchenne muscular dystrophy gene, a collagen VI alpha 1gene (COL6A1), a myotubular myopathy 1 gene (MTM1), a dysferlin gene(DYSF), a laminin-alpha 2 gene (LAMA2), an emery-dreyfuss musculardystrophy gene (EMD), and/or a Calpain 3 gene (CAPN3). The invention isfurther delineated by means of examples drawn from the Duchenne musculardystrophy gene. Although this gene constitutes a particularly preferredgene in the invention, the invention is not limited to this gene.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD)are both caused by mutations in the DMD gene that is located on the Xchromosome and codes for dystrophin (1-6). DMD has an incidence of1:3500 newborn males. Patients suffer from progressive muscle weakness,are wheelchair bound before the age of 13 and often die before the thirddecade of their life (7). The generally milder BMD has an incidence of1:20,000. BMD patients often remain ambulant for over 40 years and havelonger life expectancies when compared to DMD patients (8).

Dystrophin is an essential component of the dystrophin-glycoproteincomplex (DGC), which, amongst others, maintains the membrane stabilityof muscle fibers (9, 10). Frame-shifting mutations in the DMD generesult in dystrophin deficiency in muscle cells. This is accompanied byreduced levels of other DGC proteins and results in the severe phenotypefound in DMD patients (11, 12). Mutations in the DMD gene that keep thereading frame intact, generate shorter, but partly functionaldystrophins, associated with the less severe BMD (13, 14).

Despite extensive efforts, no clinically applicable and effectivetherapy for DMD patients has yet been developed (15), although a delayof the onset and/or progression of disease manifestations can beachieved by glucocorticoid therapy (16). Promising results have recentlybeen reported by us and others on a genetic therapy aimed at restoringthe reading frame of the dystrophin pre-mRNA in cells from the mdx mousemodel and DMD patients (17-23). By the targeted skipping of a specificexon, a DMD phenotype can be converted into a milder BMD phenotype. Theskipping of an exon can be induced by the binding of antisenseoligoribonucleotides (“AONs”), targeting either one or both of thesplice sites or exon-internal sequences. Since an exon will only beincluded in the mRNA when both the splice sites are recognized by thespliceosome complex, splice sites are obvious targets for AONs. This wasshown to be successful, albeit with variable efficacy and efficiency(17, 18, 20, 21).

We hypothesized that targeting exon-internal sequences might increasespecificity and reduce interference with the splicing machinery itself.Some exons have weak splice sites and appear to require binding of an SRprotein to an exon recognition sequence (ERS) or an exonic splicingenhancer (ESE) to be properly recognized by the splicing machinery (24).SR proteins are a highly conserved family of arginine-serine-rich,spliceosome-associated phosphoproteins essential for pre-mRNA splicing(50, 51). SR proteins appear to act early in splicing by promotingsplice site recognition and spliceosome assembly. SR proteins also playa regulatory role, because they can determine alternative splice siteusage in vivo and in vitro. SR proteins appear to be recruited fromnuclear “speckles” in which they are concentrated, to sites oftranscription in order to spatially coordinate transcription andpre-mRNA splicing within the cell nucleus (49, 52). Disruptive pointmutations or AONs that block these sequences have been found to resultin exon skipping (19, 22, 24-28). Using exon-internal AONs specific foran ERS-like sequence in exon 46, we were previously able to modulate thesplicing pattern in cultured myotubes from two different DMD patientswith an exon 45 deletion (19). Following AON treatment, exon 46 wasskipped, which resulted in a restored reading frame and the induction ofdystrophin synthesis in at least 75% of the cells. We have recentlyshown that exon skipping can also efficiently be induced in humancontrol muscle cells for 15 different DMD exons using exon-internal AONs(23, unpublished results).

In contrast to the previous opinion that skipping can only be achievedwith weak splice sites or exons containing ERS-like sequences, we haveseen that of the exons that were skipped in the invention, most do nothave weak splice sites nor do they contain ERS-like sequences. Thus,binding of the AONs to the targeted exon per se is sufficient to causeexon skipping, either by interfering with one or more components of thesplicing machinery or by altering the secondary structure of the RNA insuch a manner that the splicing machinery no longer recognizes the exon.In a preferred embodiment, the exon to be skipped comprises exons 2, 8,9, 17, 19, 29, 40-46, 49-53, 55 or 59; more preferably, exons 2, 8, 9,17, 40, 41, 42, 44, 49-52 or 59. In yet another embodiment, the exon tobe skipped comprises exons 2, 29, 40, 41, 42, 43, 44, 45, 46, 49, 50, 51or 53.

Any oligonucleotide fulfilling the requirements of the invention may beused to induce exon skipping in the DMD gene. In a preferred embodiment,an oligonucleotide comprises a sequence as depicted as active inexon-skipping in Table 2, or a functional equivalent thereof comprisinga similar, preferably the same, hybridization capacity in kind, notnecessarily in amount. Preferably, an oligonucleotide comprising asequence as depicted in Table 2, derived from the exons 2, 40, 41, 42,43, 44, 45, 49, 50, 51 or 53, demonstrably active in exon skipping.

Reading frame correction can be achieved by skipping one or two exonsflanking a deletion, by skipping in-frame exons containing a nonsensemutation, or by skipping duplicated exons. This results in proteinssimilar to those found in various EMD patients (2, 29). A survey of theLeiden DMD mutation database (WorldWideWeb.dmd.nl; (30)) evinces that wecan thus correct over 75% of DMD-causing mutations (see Table 4). Weshow the actual therapeutic effect of exon skipping for seven differentmutations. In all patient muscle cell cultures, we were able to restoredystrophin synthesis in 75% to 80% of treated cells.

The complementary oligonucleotide generated through a method of theinvention is preferably complementary to a consecutive part of between16 and 50 nucleotides of the exon RNA. Different types of nucleic acidmay be used to generate the oligonucleotide. Preferably, theoligonucleotide comprises RNA, as RNA/RNA hybrids are very stable. Sinceone of the aims of the exon skipping technique is to direct splicing insubjects, it is preferred that the oligonucleotide RNA comprises amodification providing the RNA with an additional property, forinstance, resistance to endonucleases and RNaseH, additionalhybridization strength, increased stability (for instance, in a bodilyfluid), increased or decreased flexibility, reduced toxicity, increasedintracellular transport, and/or tissue-specificity, etc. Preferably, themodification comprises a 2′-O-methyl-phosphorothioateoligoribonucleotide modification.

With the advent of nucleic acid-mimicking technology, it has becomepossible to generate molecules that have a similar, preferably the same,hybridization characteristics, in kind, not necessarily in amount, asnucleic acid itself. Such equivalents are, of course, also part of theinvention. Examples of such mimics equivalents are peptide nucleic acid,locked nucleic acid and/or a morpholino phosphorodiamidate. Suitable butnon-limiting examples of equivalents of oligonucleotides of theinvention can be found in C. Wahlestedt et al., Potent and non-toxicantisense oligonucleotides containing locked nucleic acids, Proc. Natl.Acad. Sci, U.S.A. 97:5633-8 (2000); A. N. Elayadi and D. R. Corey,Application of PNA and LNA oligomers to chemotherapy, Cuff. Opin,Investig. Drugs 2:558-61 (2001); H. J. Larsen, T. Bentin, and P. E.Nielsen, Antisense properties of peptide nucleic acid, Chem. Biophys,Acta 1489:159-66 (1999); D. A. Braasch and D. R Corey, Novel antisenseand peptide nucleic acid strategies for controlling gene expression,Biochemistry 41:4503-10 (2002); J. Summerton and D. Weller, Morpholinoantisense oligomers: design, preparation, and properties, AntisenseNucleic Acid Drug Dev. 7:187-95 (1997). Hybrids between one or more ofthe equivalents among each other and/or together with nucleic acid are,of course, also part of the invention. In a preferred embodiment, anequivalent comprises locked nucleic acid, as locked nucleic aciddisplays a higher target affinity and reduced toxicity and, therefore,shows a higher efficiency of exon skipping.

An oligonucleotide of the invention typically does not have to overlapwith a splice donor or splice acceptor of the exon.

An oligonucleotide of the invention, or equivalent thereof, may, ofcourse, be combined with other methods for interfering with thestructure of an mRNA. It is, for instance, possible to include in amethod at least one other oligonucleotide that is complementary to atleast one other exon in the pre-mRNA. This can be used to preventinclusion of two or more exons of a pre-mRNA in MRNA produced from thispre-mRNA. In a preferred embodiment, at least one other oligonucleotideis an oligonucleotide, or equivalent thereof, generated through a methodof the invention. This part of the invention is further referred to asdouble- or multi-exon skipping. In most cases, double-exon skippingresults in the exclusion of only the two targeted (complementary) exonsfrom the pre-mRNA. However, in other cases, it was found that thetargeted exons and the entire region in between the exons in thepre-mRNA were not present in the produced mRNA even when other exons(intervening exons) were present in such region. This multi-skipping wasnotably so for the combination of oligonucleotides derived from the DMDgene, wherein one oligonucleotide for exon 45 and one oligonucleotidefor exon 51 was added to a cell transcribing the DMD gene. Such a set-upresulted in mRNA being produced that did not contain exons 45 to 51.Apparently, the structure of the pre-mRNA in the presence of thementioned oligonucleotides was such that the splicing machinery wasstimulated to connect exons 44 and 52 to each other.

It has now also been found possible to specifically promote the skippingof the intervening exons by providing a linkage between the twocomplementary oligonucleotides. To this end, provided is a compoundcapable of hybridizing to at least two exons in a pre-mRNA encoded by agene, the compound comprising at least two parts, wherein a first partcomprises an oligonucleotide having at least eight consecutivenucleotides that are complementary to a first of at least two exons, andwherein a second part comprises an oligonucleotide having at least eightconsecutive nucleotides that are complementary to a second exon in thepre-mRNA. The at least two parts are linked in the compound so as toform a single molecule. The linkage may be through any means but ispreferably accomplished through a nucleotide linkage. In the lattercase, the number of nucleotides that do not contain an overlap betweenone or the other complementary exon can be zero, but is preferablybetween 4 to 40 nucleotides. The linking moiety can be any type ofmoiety capable of linking oligonucleotides. Currently, many differentcompounds are available that mimic hybridization characteristics ofoligonucleotides. Such a compound is also suitable for the invention ifsuch equivalent comprises similar hybridization characteristics in kind,not necessarily in amount. Suitable equivalents were mentioned earlierin this description. One, or preferably more, of the oligonucleotides inthe compound are generated by a method for generating an oligonucleotideof the invention. As mentioned, oligonucleotides of the invention do nothave to consist of only oligonucleotides that contribute tohybridization to the targeted exon. There may be additional materialand/or nucleotides added.

As mentioned, a preferred gene for restructuring mRNA is the DMD gene.The DMD gene is a large gene, with many different exons. Consideringthat the gene is located on the X-chromosome, it is mostly boys that areaffected, although girls can also be affected by the disease, as theymay receive a bad copy of the gene from both parents, or are sufferingfrom a particularly biased inactivation of the functional allele due toa particularly biased X chromosome inactivation in their muscle cells.The protein is encoded by a plurality of exons (79) over a range of atleast 2.6 Mb. Defects may occur in any part of the DMD gene. Skipping ofa particular exon or particular exons can very often result in arestructured mRNA that encodes a shorter than normal, but at leastpartially functional, dystrophin protein. A practical problem in thedevelopment of a medicament based on exon-skipping technology is theplurality of mutations that may result in a deficiency in functionaldystrophin protein in the cell. Despite the fact that already multipledifferent mutations can be corrected for by the skipping of a singleexon, this plurality of mutations requires the generation of a largenumber of different pharmaceuticals; as for different mutations,different exons need to be skipped.

An advantage of a compound capable of inducing skipping of two or moreexons, is that more than one exon can be skipped with a singlepharmaceutical. This property is practical and very useful in that onlya limited number of pharmaceuticals need to be generated for treatingmany different Duchenne or Becker mutations. Another option now open tothe person skilled in the art is to select particularly functionalrestructured dystrophin proteins and produce compounds capable ofgenerating these preferred dystrophin proteins. Such preferred endresults are further referred to as mild phenotype dystrophins. Thestructure of the normal dystrophin protein can be schematicallyrepresented as two endpoints having structural function (the beads),which are connected to each other by a long, at least partly flexible,rod. This rod is shortened in many Becker patients.

This led the field to the conclusion that not so much the length of therod, but the presence of a rod and the composition thereof (with respectto particular hinge regions in the protein), is crucial to the functionper se of the dystrophin protein. Though the size of the rod may have animpact on the amount of functionality of the resulting (Becker) protein,there are many notable exceptions. These exceptions will be detailedbelow. There are especially benign mutations that can have a very shortrod. It was noted by the inventors that many more different types ofBecker patients should have been detected in the patient population.However, some types of shortened dystrophin proteins, that according tothis hypothesis should have a Becker phenotype, are not detected inhuman population. For some of these “theoretical” Becker forms, thiscould just be a matter of chance. However, in the invention, it has beenfound that at least some of these “potential” Becker patients have sucha benign phenotype that subjects having these types of mutations do notpresent themselves to a doctor or are not diagnosed as suffering fromBecker's disease. With a compound of the invention, it is possible torestructure DMD pre-mRNA of many different Duchenne and even Beckerpatients such that a mild phenotype dystrophin is generated aftertranslation of the restructured mRNA.

Thus provided is a particularly preferred compound, wherein the parts ofthe compounds at least comprise a first part comprising anoligonucleotide or equivalent thereof, complementary to exon 17, and asecond part comprising an oligonucleotide or equivalent thereof,complementary to exon 48. The resulting restructured mRNA encodes anin-frame shortened dystrophin protein, lacking all exons from 17 to 48.This shortened dystrophin protein mimics a mild phenotype dystrophin asmentioned above. The compound (referred to as the 17-48 compound)should, according to current databases, be able to deal with as much as20% of the patients having a DMD mutation currently characterized.Another preferred compound is the 45-55 compound. This compound should,according to the same calculations, be able to deal with 38% of thepatients having a DMD mutation thus far characterized.

In yet another embodiment, the compound comprises a 42-55 compound or a49-59 compound capable of dealing with, respectively, 65% and 18% of thecurrently characterized DMD patients. Preferred are a 45-49 compound anda 45-51 compound, preferably in the form as disclosed in theexperimental part, having the potential to treat, respectively, 4% and8% of the DMD patients characterized thus far.

Another aspect of the invention is a compound capable of hybridizing toone exon in a pre-mRNA encoded by a gene, the compound comprising atleast two parts, wherein a first part comprises an oligonucleotide ofwhich at least a part of the oligonucleotide is complementary to theclosed structure and wherein a second part comprises an oligonucleotideof which at least part is complementary to the open structure. The openand closed structures are, of course, determined from a secondarystructure of RNA from the exon. A compound having two distinguishableparts complementary to a single exon may comprise an oligonucleotide, orequivalent thereof, or combination thereof, as mentioned herein in themethod for generating the oligonucleotide.

A transcription system containing a splicing system can be generated invitro. The art has suitable systems available. However, the need formRNA restructuring is, of course, predominantly felt for themanipulation of living cells, preferably, cells in which a desiredeffect can be achieved through the restructuring of an mRNA. PreferredmRNAs that are restructured are listed hereinabove. Preferably, genesactive in muscle cells are used in the invention. Muscle cells (e.g.,myotubes) are multinucleated cells in which many, but not all, musclecell-specific genes are transcribed via long pre-mRNA. Such longpre-mRNAs are preferred for the invention, as restructuring of MRNAsproduced from such long mRNAs is particularly efficient. It is thought,though it need not necessarily be so, that the relatively long timeneeded to generate the full pre-mRNA aids the efficiency ofrestructuring using a method or means of the invention, as more time isallowed for the process to proceed. The preferred group of genes ofwhich the mRNA is preferably restructured in a method of the inventioncomprises: COL6A1 causing Bethlem myopathy, MTM1 causing myotubularmyopathy, DYSF (dysferlin causing Miyoshi myopathy), and LGMD, LAMA2(Laminin alpha 2) causing Merosin-deficient muscular dystrophy, EMD(emerin) causing Emery-Dreyfuss muscular dystrophy, the DMD gene causingDuchenne muscular dystrophy and Becker muscular dystrophy, and CAPN3(calpain) causing LGMD2A. Any cell may be used, however, as mentioned, apreferred cell is a cell derived from a DMD patient. Cells can bemanipulated in vitro, i,e., outside the subject's body. However,ideally, the cells are provided with a restructuring capacity in vivo.Suitable means for providing cells with an oligonucleotide, equivalentor compound of the invention are present in the art. Improvements inthese techniques are anticipated considering the progress that hasalready thus far been achieved. Such future improvements may, of course,be incorporated to achieve the mentioned effect on restructuring of mRNAusing a method of the invention. At present, suitable means fordelivering an oligonucleotide, equivalent or compound of the inventionto a cell in vivo comprises polyethylenimine (PEI) or syntheticamphiphils (SAINT-18) suitable for nucleic acid transfections. Theamphiphils show increased delivery and reduced toxicity, also when usedfor in vivo delivery. Preferably, compounds mentioned in Smisterova, A.Wagenaar, M. C. A. Stuart, E. Polushkin, G. ten Bunke, R. Hulst, J. B.F. N. Engberts, and D. Hoekstra, “Molecular shape of the Cationic LipidControls the Structure of the CationicLipid/Dioleylphosphatidylethanolomine-DNA Complexes and the Efficiencyof Gene Delivery,” J. Biol. Chen. 2001, 276:47615. The syntheticamphiphils preferably used are based upon the easily syntheticallyavailable “long-tailed” pyridinium head group-based materials. Withinthe large group of amphiphils synthesized, several show a remarkabletransfection potential combined with a low toxicity in terms of overallcell survival. The ease of structural modification can be used to allowfurther modifications and the analysis of their further (in vino)nucleic acid transfer characteristics and toxicity.

An oligonucleotide, equivalent thereof, or a compound according to theinvention may be used for, at least in part, altering recognition of theexon in a pre-mRNA. In this embodiment, the splicing machinery is atleast in part prevented from linking the exon boundaries to the mRNA.The oligonucleotide, equivalent or compound of the invention is at leastin part capable of altering exon recognition in a pre-mRNA. This use isthus also provided in the invention. The prevention of inclusion of atargeted exon in an mRNA is also provided as a use for, at least inpart, stimulating exon skipping in a pre-mRNA. As mentioned above, thetargeted exon is not included in the resulting mRNA. However, part ofthe exon (a neo-exon) may occasionally be retained in the produced mRNA.This sometimes occurs when the targeted exon contains a potential spliceacceptor and/or splice donor sequence. In this embodiment, the splicingmachinery is redirected to utilize a previously unused (or underused)splice acceptor/donor sequence, thereby creating a new exon (neo-exon).The neo-exon may have one end in common with the paleo-exon, althoughthis does not always have to be the case. Thus, in one aspect, anoligonucleotide, equivalent or compound of the invention is used foraltering the efficiency with which a splice donor or splice acceptor isused by a splicing machinery.

In view of the foregoing, further provided is the use of anoligonucleotide, an equivalent thereof or a compound of the inventionfor the preparation of a medicament. Further provided is apharmaceutical preparation comprising an oligonucleotide, equivalentthereof or a compound according to the invention. An oligonucleotide, anequivalent thereof or a compound of the invention can be used for thepreparation of a medicament for the treatment of an inherited disease.Similarly provided is a method for altering the efficiency with which anexon in a pre-mRNA is recognized by a splicing machinery, the pre-mRNAbeing encoded by a gene comprising at least two exons and at least oneintron, the method comprising providing a transcription systemcomprising the splicing machinery and the gene, with an oligonucleotide,equivalent thereof or a compound according to the invention, wherein theoligonucleotide, equivalent thereof or compound is capable ofhybridizing to at least one of the exons, and allowing for transcriptionand splicing to occur in the transcription system. The gene may compriseat least three exons.

An oligonucleotide of the invention may be provided to a cell in theform of an expression vector, wherein the expression vector encodes atranscript comprising the oligonucleotide. The expression vector ispreferably introduced into the cell via a gene delivery vehicle. Adelivery vehicle may be a viral vector such as an adenoviral vector and,more preferably, an adeno-associated virus vector. Also provided aresuch expression vectors and delivery vehicles. It is within the skill ofthe artisan to design suitable transcripts. Preferred for the inventionare PolIII-driven transcripts, preferably in the form of a fusiontranscript with a Ul or U7 transcript. Such fusions may be generated asdescribed in references 53 and 54,

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F show RT-PCR and sequence analysis of dystrophin mRNAfragments of the AON-treated DMD patient myotube cultures (patient DL515.2 (1A); patient DL363.2 (1B); patient 50685.1 (1C); patient DL 589.2(1D); patient 53914.1 (1E); patient 50423.1 (1F)), focusing on theregions encompassing the exons targeted for skipping. Shorter noveltranscripts were observed when compared to the untransfected myotubecultures (NT). Sequence analysis confirmed the precise skipping of thetargeted exons. An alternatively spliced product, detected for patient50685.1 (1C) was sequenced and found to be derived from activation of acryptic splice site in exon 51. Shorter fragments, detected inuntransfected myotube cultures from DL 363.2 (1B), DL 589.2 (1D) and53914,1 (1E), were sequenced and found to be the result of thespontaneous skipping of exons 44, 50 and 53, respectively. Note that insome analyses, additional fragments, slightly shorter than the wild-typeproducts, were present. This was due to heteroduplex formation. 100 bp:size marker, -RT-PCR: negative control.

FIGS. 2A-2F illustrate immuno-histochemical analysis of the AON-treatedmyotube cultures from the six different DMD patients (patient DL 515.2(2A); patient DL363.2 (2B); patient 50685.1 (2C); patient DL 589.2 (2D);patient 53914.1 (2E); patient 50423.1 (2F)). Cells were stained formyosin to identify fully differentiated myotubes (not shown). Monoclonalantibodies MANDYS1 (middle panel) and Dys2 (right panel) were used todetect dystrophin one to four days post-transfection. No dystrophinsignals could be detected in untreated cells stained with MANDYS1 (leftpanel) nor Dys2 (not shown), whereas clear, mainly cytoplasmicdystrophin signals could be detected for each patient upon the inducedexon skipping. In patients DL 363.2 (2B), DL 589.2 (2D) and 53914.1(2E), dystrophin membrane signals could be observed. Note that membranesignals were more often found for Dys2, which recognizes the full-lengthdystrophin. MANDYS1 recognizes an internal part of dystrophin and ismore prone to generate cytoplasmic signals, since it also detectsdystrophin in the first stages of synthesis. Magnification 63×.

FIGS. 3A-3F are Western blot analyses of the AON-treated myotubecultures from six different patients (patient DL 515.2 (3A); patientDL363.2 (3B); patient 53914.1 (3C); patient 50685.1 (3D); patient DL589.2 (3E); patient 50423.1 (3F)). Monoclonal antibody DY4 was used todetect dystrophin. Protein extracts isolated from human control myotubecultures (HC) were used as a positive control (3C and 3F). To avoidover-exposure, this sample was 1 to 10 diluted. To demonstrate equalloading of protein samples, blots were additionally stained with anantibody against myosin. No, or, as a result of spontaneous exonskipping, very low (3E and 3C) levels of dystrophin were detected innon-transfected Myotube cultures (NT). Clear dystrophin signals wereobserved in AON-treated myotube cultures for each of the patients. For50685.1 and DL 363.2, a time-course experiment was performed. Dystrophincould be detected 16 hours post-transfection and was found at increasinglevels at 24 hours and 48 hours post-transfection for 50685.1 (3D). ForDL 363.2, dystrophin could be detected in increasing levels up to sevendays post-transfection (3B). For patients DL 515.2 (3A), DL 363.2 (3B)and DL 589.2 (3E), the detected dystrophin was significantly shorterthan the control dystrophin. This is due to the size of the deletions inthese patients.

FIGS. 4A-4B show immuno-histochemical analysis of four DGC proteins fromtreated myotube cultures from patient DL 363.2. Cells were stained formyosin to identify sufficiently differentiated myotubes (not shown).Monoclonal antibodies NCL-a-SARC, NCL-b-SARC, NCL-g-SARC and NCL-b-DGwere used to detect α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan andβ-dystroglycan, respectively. These proteins were detected in reducedpercentages (40%) in untreated myotubes, and were mainly located in thecytoplasm (4A). Following AON treatment, however, α-sarcoglycan wasdetected in 70%, B-sarcoglycan was detected in 90%, γ-sarcoglycan wasdetected in 90% and β-dystroglycan was detected in 80% of the myotubes,and the proteins were mostly membrane-bound (4B), Magnification 63×.

FIGS. 5A-5I are RT-PCR analyses of human dystrophin mRNA in the regionsencompassing the exons targeted for skipping. Exon skipping was assessedusing AONs directed to exon 2 (5A and 5B), exon 29 (5C), exon 40, 41 or42 (5D), exon 43, 44 or 45 (5E), exon 46 (5F), exon 47, 48, 49 or 50(5G), exon 51 (5H) and exon 53 (5I). Shorter novel transcript fragmentswere observed following transfection with the different AONs whencompared to non-transfected myotube cultures (NT). Sequence analysis(not shown) confirmed the skipping of the targeted exons, as indicatedby the labels adjacent to the images. Alternatively spliced products,detected in the regions around exon 2 (b), exon 29 (c), and exon 51 (h),were sequenced and found to be derived from either co-skipping ofadjacent exons or usage of a cryptic splice site. No specific (RT-) PCRproducts were obtained. In some analyses, additional fragments, lightlyshorter than the wild-type products, were present. This was due toheteroduplex formation.

FIG. 6 illustrates double-exon skipping in DMD patient DL90.3 carrying anonsense mutation in the out-of-frame exon 43. RT-PCR analysis ofdystrophin mRNA fragments of AON-treated myotubes from this patientshowed a shorter, novel transcript not present in untransfected myotubes(NT). Sequence analysis confirmed the precise skipping of the targetedexons 43 and 44. Besides this double-skip, we also detected asingle-exon 44 skip. Note that the additional fragment, slightly shorterthan the wild-type product, is due to heteroduplex formation. 100 bp:size marker, -RT-PCR: negative control.

FIGS. 7A-7D illustrate double- and multi-exon skipping in human controlmyotubes (KM 109), DMD) (7A) patient DL 470.2 (7B), carrying a deletionof exons 46 to 50, and DMD patient 50685,1, carrying a deletion of exons48 to 50 (7C). FIG. 7D is RT-PCR analysis of dystrophin mRNA fragmentsin the myotube cultures treated with either a mixture of h45A0N5 andh51A0N2 (mix) or with a U-linked combination of AONs (U: h45AON5 linkedto h51AON2 by ten uracil nucleotides). In all samples treated witheither the mix of AONs or the U-linker AON, a shorter transcriptfragment was detected that contained exon 44 spliced to exon 52, andthat was not present in untreated myotubes (NT). This novel, in-frametranscript arose from double-exon skipping in patient AL 470.2 (thetargeted exons 45 and 51 are directly flanking the deletion), but frommulti-exon skipping in both the human control and patient 50685.1. Inthe treated patient myotube cultures, additional shorter fragments wereobserved due to single-exon 45 and single-exon 51 skipping. Note that insome lanes, other fragments, slightly shorter than the wild-typeproducts, were present. This was due to heteroduplex formation. 100 bp:size marker, -RT-PCR: negative control. FIG. 7D shows that all fragmentswere quantified using the DNA 7500 LABCHIP® and the BIOANALYZER™(Agilent). The percentage of double- or multi-exon 45 to 51 skipping wasdetermined by the ratio of this fragment to the total of transcriptfragments. The U-combined AON seems less efficient in AL 470.2, but moreefficient in KM 109 and 50685.1, when compared to the mixture of AONs.

DETAILED DESCRIPTION Examples Example 1 Results

This study includes six DMD patients affected by different mutations(Table 1). Patient DL 515.2 carries an exon 45-50 deletion; hence exon51 skipping would be frame correcting. Patient DL 363.2 has a deletionof exon 45-54; the reading frame for this patient would be corrected byan exon 44 skip. For patient 50685.1, who is affected by an exon 48-50deletion, reading frame correction requires an exon 51 skip. Patient DL5892 has an exon 51-55 deletion; the reading frame would be corrected byan exon 50 skip. Patient 53914.1 carries a single-exon 52 deletion.Notably, in this case, both the skipping of exon 51 or exon 53 would beframe correcting. Finally, patient 50423.1 has a deletion of a singlebase pair in exon 49, at position 7389 on cDNA level, resulting in aframe-shift and a premature stop codon in exon 49. Since exon 49 is anin-frame exon, skipping of this exon would correct the reading frame forthis patient.

We have previously identified AONs with which the skipping of thementioned target exons 44, 49, 50, 51 and 53 can be induced atconcentrations of 1 μM (23). In subsequent dose-response experiments,however, we have obtained substantial skipping efficiencies with lowerconcentrations of 500 nM or 200 nM, and even 100 nM for most AONs (datanot shown). This had the extra advantageous effect of lower doses of PEIrequired for transfection, which significantly reduced the levels ofcytotoxicity as found in our earlier transfection experiments. Myotubecultures from the six DMD patients were transfected with the relevantAONs. On average, 70% to 90% of cells showed specific nuclear uptake offluorescent AONs. RNA was isolated 24 hours post-transfection andanalyzed by RT-PCR (FIGS. 1A-1F). In all patients, the targeted exonswere skipped at high efficiencies, and precisely at the exon boundaries,as confirmed by sequence analysis of the novel shorter transcripts(FIGS. 1A-1F). For patient 50685.1, an additional transcript fragmentwas found (FIG. 1C). Sequence analysis showed that this was generated bythe activation of a cryptic splice site in exon 51. This was previouslyalso observed in human control cells treated with the same AON (23).Remarkably, low levels of spontaneous exon skipping were observed inuntreated cells derived from patients DL 363.2 (exon 44 skip), DL 589.2(exon 50 skip), and 53914.1 (exon 53 skip). RT-PCR analysis on severallarger areas of the DMD gene transcript did not reveal additional,unexpected, aberrant splicing patterns induced by the AON-treatment.

The resulting in-frame transcripts should restore dystrophin synthesis.Indeed, immuno-histochemical analysis of transfected myotube culturesdetected dystrophin in the majority of myotubes for each patient (FIGS.2A-2F). The therapeutic efficiency was determined by double staining,using antibodies against myosin, to identify sufficiently differentiatedmyotubes and dystrophin. On average, 75% to 80% of myosin-positivemyotubes showed dystrophin expression. We observed clear membrane-bounddystrophin for patients DL 363.2, DL 589.2 and 53914.1 two dayspost-transfection (FIGS. 2B, 2D, and 2E). The presence of dystrophin wasconfirmed for each patient by Western blot analysis (FIGS. 3A-3F). Forpatients 50685.1 and DL 363.2, we performed time course experiments,which indicated that dystrophin can be detected as soon as 16 hourspost-transfection (FIG. 3D) and at increasing levels up to seven dayspost-transfection (FIG. 3B). The dystrophin proteins from patientsDL515.2, DL 363.2 and DL 589.2 are significantly shorter than the humancontrol, which is due to the size of the deletion.

For one patient, DL 363.2, we also assessed whether the induction of thedystrophin synthesis resulted in the restoration of the DGC (FIGS.4A-4B). Prior to AON treatment, we found reduced, mainly cytoplasmaticalpha, beta, gamma sarcoglycan and beta-dystroglycan signals (30%, 30%,40% and 80%, respectively) (FIG. 4A). Following AON transfection,increased levels of mainly membrane-bound alpha-, beta- andgamma-sarcoglycans and beta-dystroglycan were detected in 70%, 90%, 90%and 80% of the treated myotube cultures, respectively (FIG. 4B).

DISCUSSION

The reading frame correction strategy for DMD patients is aimed atantisense-induced, targeted exon skipping. This would convert a severeDMD phenotype into a mostly milder BMD phenotype. We determined thebroad applicability in six patients, carrying five different deletionsand a point mutation in an exon 49 (Table I). Following AON treatment,we show for each patient the precise skipping of the targeted exon onthe RNA level, and a dystrophin protein in 75% to 80% of the treatedmyotubes. In particular, we here report, for the first time, theapplication of a single AON treatment (i.e., the induced skipping ofexon 51) to correct the reading frame for several different deletions.

Interestingly, the levels of exon skipping observed in the DMD patientcells are significantly higher than those previously obtained in humancontrol cells (23). Typically, the novel skip transcript is the majorproduct. This can be explained by the action of the nonsense-mediateddecay (NMD) process (25, 32), In control cells, the skip of anout-of-frame exon results in an out-of-frame transcript, which will besusceptible to NMD. In patient cells, the skip of a target exon resultsin an in-frame transcript that would be resistant to NMD and thus morestable than the out-of-frame transcript originally present.

For three of the patients (DL 363.2, DL 589.2 and 53914.1), we detectedlow levels of spontaneous skipping of exons 44, 50 and 53 in untreatedcells. This phenomenon has previously also been described for so-calledrevertant muscle fibers (33-35). These dystrophin-positive fibers arepresent in low amounts (2% to 10%) in DMD muscles and are considered tobe the result of secondary somatic mutations and/or alternative splicingthat restore the reading frame, The existence of revertant fibers hasbeen suggested to correlate with the severity of the disease (36, 37)

Restoration of the dystrophin synthesis could be detected as soon as 16hours post-transfection. At two days post-transfection, dystrophin wasdetected at the membrane, indicating that these novel BMD-like proteinsare likely in part functional. Furthermore, we show that restoration ofthe dystrophin synthesis appears to re-establish the formation of thedystrophin-glycoprotein complex.

In patients DL 363.2 and DL 589.2, the targeted exon skipping enlargedthe deletions to span exons 44-54 and 50-55, respectively. So far, thesedeletions have not been reported in DMD or BMD patients. This means thatthey either do not exist or generate a very mild phenotype not diagnosedas BMD. Considering both the large variety of BMD mutations and themarkedly lower incidence of BMD observed, we consider the lastexplanation more plausible than the first. The out-of-frame deletionsfrom patients DL 515.1, 50685.1 and 50423.1 were converted into in-framedeletions as observed in BMD patients carrying deletions of exon 45-51,exon 48-51 and exon 49 (30, 38-40); noteworthy, the exon 48-51 deletionhas even been described in an asymptomatic person (40). On the otherhand, however, there are also DMD patients carrying such deletions (38,41-43). Since most of these theoretical in-frame deletions have beendetected on the DNA level only, we hypothesize that the dystrophindeficiency in these DMD patients may be caused by additional aberrantsplicing patterns on the RNA level, resulting in an out-of-frametranscript.

It is feasible to correct over 75% of the mutations reported in theLeiden DMD-mutation database (30). Our results indicate thatantisense-induced reading frame correction will be a promisingtherapeutic approach for many DMD patients carrying different deletionsand point mutations. Towards the establishment of clinical trials, weare currently investigating and optimizing delivery methods in muscletissue of mice in vivo.

Material and Methods AONs and Primers

The AONs applied (Table 1) were previously described (23). They containa 5′ fluorescein group (6-FAM), a full-length phosphorothioate backboneand 2′-O-methyl modified ribose molecules (Eurogentec, BE). To avoidinterference with the fluorescent signals of the secondary antibodies,unlabelled AONs were used for irnmuno-histochemical analyses. Primersfor RT-PCR analysis (sequences available upon request) were synthesizedby Eurogentec (BE) or by Isogen Bioscience BV (NL).

Myogenic Cell Cultures and AON Transfections

Primary human myoblasts from patients DL 515.2 (deletion exon 45-50), DL363.2 (deletion exon 45-54), 50685.1 (deletion exon 48-50), DL 589.2(deletion exon 51-55) and 53914.1 (deletion exon 52) were isolated froma muscle biopsy and cultured as described (44). Cultures were seeded incollagen pre-coated flasks and plates (Vitrogen 100, Cohesion). Myotubeswere obtained from confluent myoblast cultures, following 7 to 14 daysof serum deprivation. They were subsequently transfected usingpolyethylenimine (PEI) for three hours in low-serum medium, according tothe manufacturer's instructions (ExGen500; MBI Fermentas), and with 3.5μl. applied per μg of transfected AON. For RT-PCR analysis,concentrations of 500 nM AON were used. At this concentration, thehighest skipping levels can be obtained, albeit with moderate levels ofcell death. Because more viable myotubes are required forimmunohistochernical and western blot analysis, concentrations of 200 nMwere applied.

For patient 50423.1, who carries a point mutation in exon 49, onlyfibroblasts were available. Following infection (MOI 50-100) with anadenoviral vector containing the MyoD gene (Ad50MyoD), the fibroblastswere forced into myogenesis according to protocols described previously(45-47). Two hours post-infection, the medium was replaced by low-serummedium, and cells were incubated for eight to ten days until myotubeswere formed. Transfection conditions were identical to those describedabove.

RNA Isolation and RT-PCR Analysis

At 24 hours post-transfection, total RNA was isolated from the myotubecultures (RNA-Bee RNA isolation solvent, Campro Scientific, NL). 300 ngof total RNA was used for RT-PCR analysis using C. therm polymerase(Roche Diagnostics, NL) in a 20 reaction at 60° C. for 30 minutes,primed with different DMD gene-specific reverse primers (Table 1).Primary PCRs were performed by 20 cycles of 94° C. (40 seconds), 60° C.(40 seconds) and 72° C. (60 seconds). One μl of these reactions was thenreamplified in nested PCRs by 32 cycles of 94° C. (40 seconds), 60° C.(40 seconds) and 72° C. (60 seconds). PCR products were analyzed on 1.5%or 2% agarose gels. Noteworthy, no evidence for a significant preferencefor the amplification of shorter fragments was obtained in PCR analyseson a defined series of mixtures of known quantities of the normal andshorter transcript fragments (data not shown).

Sequence Analysis

RT-PCR products were isolated from agarose gels using the QIAQUICK® GelExtraction Kit (Qiagen). Direct DNA sequencing was carried out by theLeiden Genorne Technology Center (LGTC) using the BigDye TerminatorCycle Sequencing Ready Reaction kit (PE Applied Biosystems) and analyzedon an ABI 3700 Sequencer (PE Applied Biosystems).

Protein Isolation and Western Blot Analysis

Protein extracts were isolated from treated myotube cultures (25 cm²flasks), using 150 μl of treatment buffer (75 mM Tris-HCl pH 6.8, 15%SDS, 5% B-mercaptoethanol, 2% glycerol, 0.001% bromophenol blue), at twoto four days post-transfection, depending on the survival rate of themyotubes. For the time course experiments, protein extracts wereisolated 4 hours, 8 hours, 16 hours, 24 hours and 48 hourspost-transfection (for patient 50685.1) or at 2 days, 4 days and 7 dayspost-transfection (for patient DL 3632).

Polyacrylamide gel electrophoresis and Western blotting were performedas described by Anderson et al., with some minor adjustments (48).Briefly, samples (75 μl) were run overnight at 4° C. on a 4% to 7%polyacrylamide gradient gel. Gels were blotted to nitrocellulose forfive to six hours at 4° C. Blots were blocked for one hour with 5%non-fat dried milk in TBST buffer (10 mM Tris-HCl, 0.15 M NaCl, 0.5%Tween 20, pH 8), followed by an overnight incubation with NCL-DYS2(which recognizes dystrophin) diluted 1:50, HRP-conjugated anti-mouse(Santa Cruz) diluted 1:10,000 was used as a secondary antibody.Immuno-reactive bands were visualized using Lumi-Lightplus WesternBlotting Substrate and scanned with a Lumi-Imager (Roche Diagnostics,NL).

Immunohistochemical Analysis

Treated myotube cultures were fixed in −20° C. methanol at one to fourdays post-transfection, depending of the survival rate of the myotubes.Prior to reaction with the different antibodies, the oncells wereincubated for one hour in a blocking solution containing 5% horse serum(Gibco BRL) and 0.05% Tween-20 (Sigma) in PBS (Gibco BRL). Allantibodies used were diluted in this blocking solution. The followingantibodies were applied: desmin polyclonal antibody (ICN Biomedicals)diluted 1:100, myosin monoclonal antibody diluted 1:100 (MF20;Developmental Studies Hybridoma Bank, University of Iowa), myosinpolyclonal antibody L53 diluted 1:100 (a gift from Dr. M. van den Hoff,AMC, NL), MANDYS1 (a gift from Dr. G, Morris, North East WalesInstitute, UK) diluted 1:10 and NCL-DYS2 (Novacastra Laboratories Ltd)diluted 1:10 to detect dystrophin, NCL-a-SARC (Novacastra LaboratoriesLtd) diluted 1:75, NCL-b-SARC (Novacastra Laboratories Ltd) diluted1:50, NCL-g-SARC (Novacastra Laboratories Ltd) diluted 1;50 and NCL-b-DG(Novacastra Laboratories Ltd) diluted 1:50 to detect α-sarcoglycan,β-sarcoglycan, γ-sarcoglycan and β-dystroglycan, respectively. After onehour incubation, slides were rinsed and incubated for one hour with thesecondary antibodies Alexa Fluor 594 goat anti-rabbit conjugate diluted1:1000 or Alexa Fluor 488 goat anti-mouse conjugate diluted 1:250(Molecular Probes Inc). The slides were analyzed using a Leica confocalmicroscope equipped with epifluorescence optics. Digital images werecaptured using a CCD camera (Photometrics).

Example 2 Materials and Methods AONs and Primers

A series of AONs (two per exon, see Table 2) was designed to bind toexon-internal target sequences showing a relatively high purine-contentand, preferably, an open secondary pre-mRNA structure (at 37° C.), aspredicted by the RNA mfold version 3.1 server [22]. The AONs varied inlength between 15 and 24 bp, with G/C contents between 26 and 67%. Theywere synthesized with the following chemical modifications: a51-fluorescein group (6-FAM), a full-length phosphorothioate backboneand 21-O-methyl-modified ribose molecules (Eurogentec, BE). The primersused for reverse transcription-polymerase chain reaction (RT-PCR)analysis (Table 3) were synthesized by Eurogentec (BE) or by IsogenBioscience BV (NL).

In Vitro Experiments

Primary human myoblasts were isolated from a muscle biopsy from anon-affected individual (KM108) by enzymatic dissociation. Briefly, thetissue was homogenized in a solution containing 5 mg/ml collagenase typeVIII (Sigma), 5 mg/ml bovine albumin fraction V (Sigma), 1% trypsin(Gibco BRL) in PBS (Gibco BRL). Following serial incubation steps of 15minutes at 37° C., suspensions containing the dissociated cells wereadded to, and pooled in, an equal volume of proliferation medium(Nut.Mix F-10 (HAM) with GlutaMax-1, Gibco BRL) supplemented with 20%fetal bovine serum (Gibco BRL) and 1% penicillin/streptomycin solution(Gibco BRL), After centrifugation, the cells were plated and furthercultured in proliferation medium, using flasks that were pre-coated withpurified bovine dermal collagen (Vitrogen 100; Cohesion).

The myogenic cell content of the culture, as determined by thepercentage of desmin-positive cells in an immunohistoehemical assay, wasimproved to 58% by repetitive pre-plating [23]. Myotubes were obtainedfrom confluent myoblast cultures following 7 to 14 days of incubation inlow-serum medium (DMEM (Gibco BRL), supplemented with 2% GlutaMax-1, 1%glucose, 2% fetal bovine serum and 1% penicillin/streptomycin solution).For transfection of the myotube cultures, we used polyethylenimine (PEI;Ex. Gen 500) according to the manufacturer's instructions (MBIFerrnentas). The cultures were transfected for three hours in low-serummedium with 1 mM of each AON linked to PEI at a ratio-equivalent of 3.5.

RNA isolation and RT-PCR analysis at 24 hours post-transfection, totalRNA was isolated from the myotube cultures using RNAzol B according tothe manufacturer's instructions (Campro Scientific, NL). One microgramof RNA was then used for RT-PCR analysis using C. therm polymerase(Roche Diagnostics) in a 20 μl reaction at 60° C. for 30 minutes, primedwith different DMD gene-specific reverse (RT) primers (Table 3). PrimaryPCRs were carried out with outer primer sets (see Table 3), for 20cycles of 94° C. (40 seconds), 60° C. (40 seconds), and 72° C. (90seconds). One microliter of this reaction was then reamplified in nestedPCRs using the appropriate primer combinations (Table 3) for 32 cyclesof 94° C. (40 seconds), 60° C. (40 seconds), and 72° C. (60 seconds).PCR products were analyzed on 1.5 or 2% agarose gels.

Sequence analysis RT-PCR products were isolated from agarose gels usingthe QIAQUICK® Gel Extraction kit (Qiagen). Direct DNA sequencing wascarried out by the Leiden Genome Technology Center (LGTC) using theBIGDYE® Terminator Cycle Sequencing Ready Reaction kit (PE AppliedBiosystems), and analyzed on an ABI 3700 Sequencer (PE AppliedBiosystems).

Results In Vitro Exon Skipping

AONs were empirically analyzed for the induction of exon skippingfollowing transfection into human control myotube cultures, using thecationic polymer polyethylenimine (PEI). As determined by the nuclearuptake of the fluorescent AONs, average transfection efficiencies of60-80% were obtained. At 24 hours post-transfection, transcripts wereanalyzed by RT-PCR using different primer combinations encompassing thetargeted exons (Table 3). Of the 30 AONs tested, a total of 21 (70%)reproducibly generated shorter transcript fragments with sizescorresponding to the specific skipping of the targeted exons (FIG. 5A-1and Table 2). In fact, as confirmed by sequence analysis of the shortertranscripts (data not shown), we could induce the specific skipping of13 out of the 15 exons targeted (five out of the seven in-frame exons,and eight out of the eight out-of-frame exons). No skipping of exons 47and 48 was detected (FIGS. 5E and G).

In the specific transcript regions that were screened in theseexperiments, we, observed in the non-transfected control myotubes,alternative splicing patterns around exons 2 and 29 (FIGS. 5B and C).The alternative products were sequenced and found to be due to theskipping of exons 2-7 (in-frame), exons 3-7 (out-of-frame), exons 28-29(in-frame), and exons 27-29 (in-frame). This genuinely occurring exonskipping was also detected previously in human skeletal muscle [24, 25].Remarkably, the level of the alternative splicing was significantlyenhanced by the AON treatment of the transfected myotube cultures. Alsonoteworthy is the observation that h2AON1 not only induced exon 2skipping in the normal transcript, but also in one of the alternativetranscripts consisting of exons 1 and 2 spliced to exon 8 (FIG. 5B).

The majority of AONs induced the precise skipping of the targeted exons,using the original splice sites of the adjacent exons. However, inresponse to h51AON2, an in-frame cryptic splice site was used in exon 51(FIG. 5H). The level of this alternatively spliced product was variablein serial transfection experiments. Finally, in some of the transfectionexperiments, additional aberrant splicing fragments were detected due tothe co-skipping of adjacent exons. Their incidence, however, wasinconsistent, and at very low levels.

References to Example 2 (Numbering in this Part Refers Strictly toNumbering Used in Example 2)

-   [1] Hoffman E. P., R H, Brown Jr., and L. M. Kunkel. Dystrophin: the    protein product of the Duchenne muscular dystrophy locus. Cell 1987,    51:919-928.-   [2] Monaco A. P., C. J. Bertelson, S. Liechti-Gallati, H. Moser,    and L. M. Kunkel. An explanation for the phenotypic differences    between patients bearing partial deletions of the DMD locus.    Genotmics 1988, 2:90-95.-   [3] Koenig M, A. H. Beggs, and M. Moyer, et al. The molecular basis    for Duchenne versus Becker muscular dystrophy: correlation of    severity with type of deletion. Am, J. Hum. Genet, 1989, 45:498-506.-   [4] Zubrzycka-Gaam E. E.; D. E. Bulman, and G. Karpati, et al. The    Duchenne muscular dystrophy gene product is localized in sarcolemma    of human skeletal muscle. Nature 1988, 333:466-469.-   [5] Yoshida M. and E. Ozawa, Glycoprotein complex anchoring    dystrophin to sarcolemma. J. Biochem, (Tokyo) 1990, 108:748-752.-   [6] Ervasti J. M. and K. P. Campbell. Membrane organization of the    dystrophin-glycoprotein complex. Cell 1991, 66:1121-1131.-   [7] Koenig M., A. P. Monaco and LM. Kunkel. The complete sequence of    dystrophin predicts a rod-shaped cytoskeletal protein. Cell 1988,    53:219-226.-   [8] van Deutekom J. C., S. S. Floyd and D. K. Booth, et al.    Implications of maturation for viral gene delivery to skeletal    muscle. Neuromuscul. Disord. 1998, 8:135-148.-   [9] Mayeda A., Y. Hayase, H. Inoue, E. Ohtsuka and Y. Ohshima.    Surveying cis-acting sequences of pre-mRNA by adding antisense    20-O-methyl oligoribonucleotides to a splicing reaction. J. Biochem.    (Tokyo) 1990, 108:399-405.-   [10] Galderisi U., A. Cascino and A. Giordano. Antisense    oligonucleotides as therapeutic agents, J. Cell. Physic!. 1999,    181:251-257.-   [11] Baker 13. F. and B. P. Monia. Novel mechanisms for    antisense-mediated regulation of gene expression. Biochim. Biophys.    Acta 1999, 1489:348.-   [12] Kole R. and R Sazani. Antisense effects in the cell nucleus:    modification of splicing. Cum Opin, Mol, Ther. 2001, 3:229-234.-   [13] Sicinski P., Y. Geng, A. S. Ryder-Cook, E. A. Barnard, M. G.    Daxlison and P. J. Barnard. The molecular basis of muscular    dystrophy in the mdx mouse: a point mutation. Science 1989,    244:15784580.-   [14] Dunckley M G., M. Manoharan, P. Villiet, LC. Eperon and G.    Dickson. Modification of splicing in the dystrophin gene in cultured    Mdx muscle cells by antisense oligoribonucleotides. Hum, Mal. Genet.    1998, 7:1083-1090.-   [15] Mann C. J., K. Honeyman and Al. Cheng, et al. Antisense-induced    exon skipping and synthesis of dystrophin in the mdx mouse. Proc.    Natl, Acad. Sci. U.S.A. 2001, 98:42-47.-   [16] Wilton S. D., F. Lloyd and K. Carville, et al. Specific removal    of the nonsense mutation from the mdx dystrophin mRNA using    anti-sense oligonucleotides. Neuromuscul. Disord, 1999, 9:330-338.-   [17] Takeshima Y., H. Wada, M. Yagi, et al. Oligonucleotides against    a splicing enhancer sequence led to dystrophin production in muscle    cells from a Duchenne muscular dystrophy patient, Brain Dev. 2001,    23:788-790.-   [18] Pramono Z. A., Y. Takeshima, H. Alimsardjono, A. Ishii, S.    Takeda and M. Matsuo. Induction of exon skipping of the dystrophin    transcript in lymphoblastoid cells by transfecting an antisense    oligodeoxynucleotide complementary to an exon recognition sequence.    Biochem. Blophys. Res. Commun, 1996, 226:445-449.-   [19] Watakabe A., K. Tanaka and Y. Shimura. The role of exon    sequences in splice site selection. Genes Dev. 1993, 7:407-418.-   [20] Tanaka K., A. Watakabe and Y. Shimura. Polypurine sequences    within a downstream exon function as a splicing enhancer. Mol. Cell    Biol. 1994, 14:1347-1354.-   [21] van Deutekom J. C., M. Bremmar-Bout, A. A. Janson, et al.    Antisense-induced exon skipping restores dystrophin expression in    DMD patient-derived muscle cells. Hum, Mol. Genet. 2001,    10:1547-1554.-   [22] Mathews D E., J. Sabina, M. Zuker and D. H. Turner. Expanded    sequence dependence of thermodynamic parameters improves prediction    of RNA secondary structure. A. J. Mol. Biol. 1999, 288:911-940.-   [23] Richter C. and D. Yaffe. The in vitro cultivation and    differentiation capacities of myogenic cell lines. Dev. Biol. 1970,    23:1-22.-   [24] Surono A., Y. Takeshima, T. Wibawa, Z. A. Pramono and M.    Mafsuo. Six novel transcripts that remove a huge intron ranging from    250 to 800 kb are produced by alternative splicing of the 50 region    of the dystrophin gene in human skeletal muscle. Biochem. Biophys.    Res. Commun. 1997, 239:895-899.-   [25] Shiga N., Y. Takeshima, H. Sakamoto, et al. Disruption of the    splicing enhancer sequence within exon 27 of the dystrophin gene by    a nonsense mutation induces partial skipping of the exon and is    responsible for Becker muscular dystrophy. J. Clin. Invest. 1997,    100:2204-2210.-   [26] Wells D. S, KB. Wells, EA. Asante, et al. Expression of human    full-length and minidystrophin in transgenic mdx mice: implications    for gene therapy of Duchenne muscular dystrophy. Hum. Mol. Genet.    1995, 4:1945-1250.-   [27] Sironi M., U. Pozzoli, R, Cagliani, G. P. Comi, A. Barden'    and N. Bresolin. Analysis of splicing parameters in the dystrophin    gene: relevance for physiological and pathogenetic splicing    mechanisms. Hum. Genet, 2001, 109:73-84.-   A. Aartsma-Rus et al., Neuromuscular Disorders 12(2002) 871-S77.

Example 3 Results Double-Exon Skipping in Two DMD Patients

This study includes two DMD patients affected by different framedisrupting mutations in the DMD gene that require the skip of two exonsfor correction of the reading frame (Table 5). Patient DL 90.3 carries anonsense mutation in exon 43. Considering that this single exon isout-of-frame, the skipping of exon 43 would remove the nonsense mutationbut not restore the reading frame.

Since the combination with exon 44 is in-frame, in this patient, weaimed at double-exon skipping, targeting both these exons. Patient DL470.2 is affected by a deletion of exons 46 to 50. Frame restorationwould require a double-exon skipping of both exons flanking thedeletion. Myotube cultures from both patients were transfected with amixture of exon 43- and 44-specific AONs (DL90.3) or exon 45- and51-specific AONs (DL470.2). The individual AONs (Table 5) werepreviously highly effective in single-exon skipping. Transfectionefficiencies were typically over 80%, as indicated by the number ofcells with specific nuclear uptake of the fluorescent AONs. RT-PCRanalysis at 24 to 48 hours post-transfection, indeed demonstrated thefeasibility of specific double-exon skipping in both samples (FIGS. 6and 7A-C). This was confirmed by sequence analysis (data not shown).Additional shorter transcript fragments were obtained due to single-exonskipping: in patient DL90.3, exon 44 skipping (FIG. 6), and in patientDL470.2, exon 51 skipping (FIG. 7B).

Multi-Exon Skipping

The splicing of exon 44 directly to exon 52 (as induced in DL470.2)generates an in-frame transcript. We hypothesized that by inducing theskipping of the entire stretch of exons in between, i.e., multi-exonskipping, we would induce a BMD-like deletion (45-51) that covers andrestores several known, smaller, DMD mutations. This would furtherenlarge the group of DMD patients that would benefit from one type offrame correction. The feasibility of multi-exon skipping was first shownin human control myotubes that were treated with a mixture of the exon45- and 51-specific AONs (FIG. 7A; KM 109). We then applied it tomyotubes from a third DMD patient carrying an exon 48-50 deletion(50685.1). By the AON-induced skipping of the (remaining) stretch ofexons in between and including exons 45 and 51, we obtained theanticipated smaller in-frame transcript with exon 44 spliced to exon 52(FIG. 7C).

Double- and Multi-Exon Skipping Using a U-Linked AON Combination

The skipping of more than one exon from one pre-mRNA molecule requiresthat both AONs are present in the same nucleus, targeting the samemolecule. To enlarge this chance, we here studied the feasibility of onecombined AON carrying both AONs specific for exons 45 and 51 (h45A0N5and h51A0N2) linked by ten uracil nucleotides (Table 5). Followingtransfection of this “U-linker AON” into myotubes from human control andthe DMD patients DL470.2 and 50685.1, RT-PCR analysis demonstrated itsefficacy to generate the anticipated in-frame transcript with exon 44spliced to exon 52 (FIGS. 7B-7C). This multi-exon skipping occurredspecifically and precisely at the exon boundaries as confirmed bysequence analysis (data not shown). In contrast to patient DL 470.2, theU-linker AON was a slightly more efficient than the mixture of AONs inthe human control and in patient 50685.1.

Materials and Methods AONs and Primers

AONs (Table 5) targeting exons 43, 44 and 51 were previously described(Aartsma-Rus, 2002). AONs targeting exon 45 were newly designed(sequences upon request). All AONs contain a 5′ fluorescein group(6-FAM), a full-length phosphorothioate backbone and 2′-O-methylmodified ribose molecules (Eurogentec, BE). To avoid interference withthe fluorescent signals of the secondary antibodies, unlabelled AONswere used for immuno-histochemical analyses. Primers for RT-PCR analysis(Table 5, sequences available upon request) were synthesized byEurogentec (BE).

RNA Isolation and RT-PCR Analysis

At 24 to 48 hours post-transfection, total RNA was isolated from themyotube cultures (RNA-Bee RNA isolation solvent, Campro Scientific, NL).300 ng of total RNA were used for RT-PCR analysis using C. therm.polymerase (Roche Diagnostics, NL) in a 20 μl reaction at 60° C. for 30minutes, primed with different DMD gene-specific reverse primers (Table5). Primary PCRs were performed by 20 cycles of 94° C. (40 seconds), 60°C. (40 seconds) and 72° C. (60 seconds). One μl of these reactions wasthen re-amplified in nested PCRs by 32 cycles of 94° C. (40 seconds),60° C. (40 seconds) and 72° C. (60 seconds), PCR products were analyzedon 1.5% or 2% agarose gels. For quantification of the transcriptproducts, nested PCRs were performed using 24 cycles. PCR products wereanalyzed using the DNA 7500 LabChip® Kit and the Agilent 2100Bioanalyzer (Agilent Technologies, NL).

Sequence Analysis

RT-PCR products were isolated from agarose gels using the QlAquick GelExtraction Kit (Qiagen). Direct DNA sequencing was carried out by theLeiden Genome Technology Center (LGTC) using the BigDye Terminator CycleSequencing Ready Reaction kit (PE Applied Biosystems) and analyzed on anABI 3700 Sequencer (PE Applied Biosystems).

Example 4 Expression Vectors Encoding a Transcript Comprising anOligonucleotide of the Invention

Due to the defined turnover rate of both the dystrophin pre-mRNA and theAONs, our DMD frame-correction therapy would require repetitiveadministrations of AONs. In addition, relatively high levels ofantisense RNA will be necessary within the nucleus, where transcriptionand splicing of the dystrophin pre-mRNA occur. Therefore, we have set upa vector system in which specific AON sequences are incorporated into amodified gene. In this example, this embodiment is described for U7small nuclear RNA (U7snRNA). U7snRNA is the RNA component of the U7ribonucleoprotein particle (U7snRNP) that is involved in the processingof the 3′ end of histone pre-mRNAs. Inherent to its function, U7snRNA isefficiently transported back from the cytoplasm to the nucleus in whichit gets subsequently incorporated into very stable U7snRNP complexes. Asimilar approach was successfully applied in AON-based gene therapystudies on B-thalassemia (53, 54). In these studies, different plasmidswere engineered containing a modified U7snRNA gene from which thenatural antisense sequence directed to the histone pre-rnRNA wasreplaced with antisense sequences targeted to differentB-thalassemia-associated aberrant splicing sites in the B-globin gene.Following transfection of these plasmids, correct splicing andexpression of the full-length B-globin protein could be restored with anefficiency of up to 65% in cultured cells expressing the differentmutant B-globin genes,

Various U7snRNA gene constructs were engineered as described inreference 53 with the modification that the B-globin sequences wereexactly replaced by the antisense sequences derived from the differentAONs. In this Example, the sequences were replaced by the antisensesequences of m46AON4, 6, 9, or 11 that were effective in inducing theskipping of mouse exon 46. A sense construct was included as negativecontrol (m46SON6). Following construct validation by sequencing, theplasmids were tested in vitro by transfection into cultured C2C12 mousemyoblasts. The U7snRNA-m46AON6 construct was most efficient.

To enhance delivery of the AON-U7snRNA gene constructs, we have clonedthem into recombinant adeno-associated viral (rAAV) vectors. AAV is asingle-stranded DNA parvovirus that is non-pathogenic and shows ahelper-dependent life cycle. In contrast to other viruses (adenovirus,retrovirus, and herpes simplex virus), rAAV vectors have demonstrated tobe very efficient in transducing mature skeletal muscle. Becauseapplication of rAAV in classical DMD “gene addition” studies has beenhindered by its restricted packaging limits (<5 kb), we applied rAAV forthe efficient delivery of the much smaller U7snRNA antisense constructs(<600 bp) to mature murine skeletal muscle. The rAAV-U7-AON vectors alsocontain the gene for green fluorescence protein (GFF-cDNA), which allowsanalysis of transduction efficiencies in muscle post-injection. Hightiter virus productions were effective in inducing exon skipping.

References (to the General Part, Excluding Example 2)

-   1. Hoffman E. P, R. H. Brown Jr., and L. M. Kunkel (1987)    Dystrophin: the protein product of the Duchenne muscular dystrophy    locus. Cell 51:919-928.-   2. Hoffman E. P, KH. Fischbeck, RH. Brown, M. Johnson, R    Medori, J. D. Loike, J. B. Harris, R. Waterston, M. Brooke, L.    Specht, et al. (1988) Characterization of dystrophin in    muscle-biopsy specimens from patients with Duchenne's or Becker's    muscular dystrophy. N. Engl. J. Med. 318:1363-1368.-   3. Den Dunnen J. T., P. M. Grootscholten, E. Bakker, L. A.    Blonden, H. B. Ginjaar, Wapenaar, H. M van Paassen, C. van    Broeckhoven, P. L. Pearson, and G. J. van ommen (1989) Topography of    the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA analysis    of 194 cases reveals 115 deletions and 13 duplications. Am. J. Hum.    Genet. 45:835-847.-   4. Koenig M., A. H. Beggs, M. Moyer, S. Scherpf, K. Heindrich, T.    Bettecken, G. Meng, C. R. Muller, M. Lindlof, H Kaariainen, et    al, (1989) The molecular basis for Duchenne versus Becker muscular    dystrophy: correlation of severity with type of deletion. Am. J.    Hum. Genet. 45:498-506.-   5. Tuffery-Giraud S., S. Chambert, J. Demaille, and M.    Claustres (1999) Point mutations in the dystrophin gene: evidence    for frequent use of cryptic splice sites as a result of splicing    defects. Hum. Mutat. 14:359-368.-   6. Prior T. W., C. Bartolo, D. K. Pearl, A C, Papp, P J.    Snyder, M. S. Sedra, A. H. Burghes, and JR. Mendell (1995) Spectrum    of small mutations in the dystrophin coding region. Am. J. Hum.    Genet. 57:22-33.-   7. Moser H. (1984) Duchenne muscular dystrophy: pathogenetic aspects    and genetic prevention. Hum. Genet. 66:17-40.-   8. Emery A. B. (2002) The muscular dystrophies. Lancet 359:687-695.-   9. Yoshida M. and E. Ozawa (1990) Glycoprotein complex anchoring    dystrophin to sarcolemma. J. Biochem. (Tokyo), 108:748-752.-   10. Ervasti J. M. and K. P. Campbell (1991) Membrane organization of    the dystrophin-glycoprotein complex. Cell 66:1121-1131.-   11. Di Blasi C., L. Moran. di, R. Barresi, F. Blasevich, F. Comelio,    and M. Mora (1996) Dystrophin-associated protein abnormalities in    dystrophin-deficient muscle fibers from symptomatic and asymptomatic    Duchenne/Becker muscular dystrophy carriers. Acta Neuropathol.    (Berl), 92:369-377.-   12. Ervasti J. M., K. Ohlendieck, S. D. Kahl, M. G. Gayer, and K. P.    Campbell (1990) Deficiency of a glycoprotein component of the    dystrophin complex in dystrophic muscle. Nature 345:315-319.-   13. Matsumura K, All. Burghes, M. Mora, F. M. Tome, L. Morandi, F.    Cornell°, F. Leturcq, M. Jeanpierre, J. C. Kaplan, P. Reinert, et    al. (1994) Immunohistochemical analysis of dystrophin-associated    proteins in Becker/Duchenne muscular dystrophy with huge in-frame    deletions in the NH2-terminal and rod domains of dystrophin. J.    Clin. Invest. 93:99-105.-   14. Monaco A R, C. J. Bertelson, S. Liechti-Gallati, H. Moser, L. M.    Kunkel (1988) An explanation for the phenotypic differences between    patients bearing partial deletions of the DMD locus. Genomics    2:90-95.-   15. Clemens P. R. and PI Duncan (2001) Progress in gene therapy for    Duchenne muscular dystrophy. Curr. Neurol. Neurosci, Rep. 1:89-96.-   16. Khan M. A. (1993) Corticosteroid therapy in Duchenne muscular    dystrophy. J. Neurol. Sci, 120:8-14.-   17. De Angelis F. G., O. Sthandier, B. Berarducci, S. Toso, G.    Galluzzi, E. Ricci, G. COSSII and I. Bozzoni (2002) Chimeric snRNA    molecules carrying antisense sequences against the splice junctions    of exon 51 of the dystrophin pre-mRNA induce exon skipping and    restoration of a dystrophin synthesis in Delta 48-50 DMD cells.    Proc. Natl. Acad. Sci. USA 99:9456-94.61.-   18. Mann C. J., K. Honeyman, A. J. Cheng, T. Ly, F. Lloyd, S.    Fletcher, J. E. Morgan, T. A. Partridge and SD. Wilton (2001)    Antisense-induced exon skipping and synthesis of dystrophin in the    mdx mouse. Proc. Natl. Acad. Sci. USA, 98:42-47.-   19. van Deutekom J. C., M. Bremmer-Bout, A. A. Janson, I. B.,    Ginjaar, F. Baas, J. T. den Dunnen and G. J. van Ommen (2001)    Antisense-induced exon skipping restores dystrophin expression in    DMD patient-derived muscle cells. Hum. Mol. Genet. 10:1547-1554.-   20. Wilton S. D., F. Lloyd, K Carville, S. Fletcher, K Honeyman, S.    Agrawal and R. Kole (1999) Specific removal of the nonsense mutation    from the mdx dystrophin mRNA using antisense oligonucleotides.    Neuromuscul. Disord. 9:330-338.-   21. Dunckley M G., M. Manoharan, P. Villiet, I. C. Eperon and G.    Dickson (1998) Modification of splicing in the dystrophin gene in    cultured Mdx muscle cells by antisense oligoribonucleotides. Hum.    Mol. Genet. 7:1083-1090.-   22. Takeshima Y., H Wada, M Yagi, Y. Ishikawa, R Minami, H. Nakamura    and M. Matsu (2001) Oligonucleotides against a splicing enhancer    sequence led to dystrophin production in muscle cells from a    Duchenne muscular dystrophy patient. Brain Dev, 23:788-790.-   23. Aartsma-Rus A., M. Bremmer-Bout, A. Janson, J. den Dunnen G, van    Ommen and J. van Deutekom (2002) Targeted exon skipping as a    potential gene correction therapy for Duchenne muscular dystrophy.    Neuromuscul. Disord. 12, S71.-   24. Shiga N., Y. Takeshima, H. Sakamoto, K Inoue, Y. Yokota, M.    Yokoyama and M. Matsuo (1997) Disruption of the splicing enhancer    sequence within exon 27 of the dystrophin gene by a nonsense    mutation induces partial skipping of the exon and is responsible for    Becker muscular dystrophy. J. Clin. Invest. 100:2204-2210.-   25. Cartegni L. S. L. Chew and A R Krainer (2002) Listening to    silence and understanding nonsense: exonic mutations that affect    splicing. Nat. Rev. Genet. 3:285-298,-   26. Schaal T. D. and T. Mafflatis (1999) Multiple distinct splicing    enhancers in the protein-coding sequences of a constitutively    spliced pre-mRNA. Mol. Cell. Blot 19:261-273.-   27. Takeshima Y., H. Nishio, H. Saka.moto, H. Nakamura and M.    Matsuo (1995) Modulation of in vitro splicing of the upstream intron    by modifying an intra-exon sequence which is deleted from the    dystrophin gene in dystrophin Kobe. J. Main Invest. 95:515-520.-   28. Pramono Z. A., Y. Takeshima, H. Alimsardjono, A. Ishii, S.    Takeda, and M. Matsu (1996) Induction of exon skipping of the    dystrophin transcript in lymphoblastoid cells by transfecting an    antisense oligodeoxynueleotide complementary to an exon recognition    sequence. Biochem. Biophys. Res. Commun. 226:445-449.-   29. Koenig M., A. P. Monaco and L. M Kunkel (1988) The complete    sequence of dystrophin predicts a rod-shaped eytoskeletal protein.    Cell 53:219-226.-   30. Den Dunnen J. (1996) The Leiden Muscular Dystrophy pages;    http://www.dmd.nl.-   31. Mann C J. K. Honeyman, G. McCloroy, S. Fletcher and S. D.    Wilton (2002) Improved antisense oligonucleotide-induced exon    skipping in the mdx mouse model of muscular dystrophy. J. Gene Med.    4:644-654.-   32. Kerr T. P., C. A. Sewry, S. A. Robb and R. G. Roberts (2001)    Long mutant dystrophins and variable phenotypes: evasion of    nonsense-mediated decay? Hum. Genet. 109:402-407.-   33. Klein C. J. D. D. Coovert, D. E. Bulman, P. N. Ray, J. R.    Mendell and A. H. Burghes (1992) Somatic reversion/suppression in    Duchenne muscular dystrophy (DMD): evidence supporting a    frame-restoring mechanism in rare dystrophin-positive fibers. Am. J.    Hum. Genet. 50; 950-959.-   34. Sherratt T. G., T. Vulliamy, V. Dubowitz, C A. Sewry and P. N.    Strong (1993) Exon skipping and translation in patients with    frameshift deletions in the dystrophin gene. Am. J. Hum. Genet.    53:1007-1015.-   35. Lu Q. L., G. E. Morris, S. D. Wilton, T. Ly, O. V.    Artem'yeva, P. Strong and T. A Partridge (2000) Massive    idiosyncratic exon skipping corrects the nonsense mutation in    dystrophic mouse muscle and produces functional revertant fibers by    clonal expansion. J. Cell Biol. 148:985-996.-   36. Nicholson L. V., M A. Johnson, K M Bushby and D.    Gardner-Medwin (1993) Functional significance of dystrophin-positive    fibers in Duchenne muscular dystrophy. Arch. Dis. Child 68:632-636,-   37. Vainzof M., M. R Passos-Bueno, R. I. Takata, C. Pavanelio Rde    and M. Zatz (1993) Intrafamilial variability in dystrophin abundance    correlated with difference in the severity of the phenotype. J.    Neurol, Sci. 119:38-42.-   38. Singh V., S. Shina, S. Mishra, L. S. Chaturvedi, S.    Pradhan, R. D. Mittal and B. Mittal (1997) Proportion and pattern of    dystrophin gene deletions in north Indian Duchenne and Becker    muscular dystrophy patients. Hum. Genet. 99:206-208.-   39. Melacini P., M. Fanin, G. A Daniell, G. Fasoli, C. Villanova, C.    Angelini, L. Vitiello, M Miorelli, G. F. Buja, M. L. Mostaccitrolo,    et al, (1993) Cardiac involvement in Becker muscular dystrophy. J.    Am, Coll. Cardiol, 22:1927-1934.-   40. Melis M A., M. Cau, F. Muntoni, A. Mateddu, R Galanello, L.    Boccone, F. Deidda, D. Loi and A Cao (1998) Elevation of serum    creatine kinase as the only manifestation of an intragenic deletion    of the dystrophin gene in three unrelated families. Europ. J.    Paediatr, Neurol. 2:255-261.-   41. Onengut, S., GN. Kavaslar, E. Battaloglu, P. Serdaroglu, F.    Deymeer, C. Ozdernir, F. Calafell and A. Tolun (2000) Deletion    pattern in the dystrophin gene in Turks and a comparison with    Europeans and Indians. Ann. Hum. Genet. 64:33-40.-   42. Rosenberg C., L. Navajas, D. F. Vagenas, E. Bakker, M.    Vainzof, M. R. Passos-Bueno, Rd, Takata, G. J. Van Ommen, M Zatz    and J. T. Den Dunnen (1998) Clinical diagnosis of heterozygous    dystrophin gene deletions by fluorescence in situ hybridization.    Neuromuscul. Disord 8:447-452.-   43. Sertic J., N. Barisic, M. Sostarko, Z. Brzovic and A.    Stavljenic-Rukavina (1997) Deletion screening of the Ducherme/Becker    muscular dystrophy gene in Croatian population, Coll. Anthropol.    21:151456.-   44. Rando T. A. and H. M. Blau (1994) Primary mouse myoblast    purification, characterization, and transplantation for    cell-mediated gene therapy. J. Cell Biol. 125:1275-1287.-   45. Murry C E., M. A. Kay, T. Bartosek, S D. Hauschka and S. M,    Schwartz (1996) Muscle differentiation during repair of myocardial    necrosis in rats via gene transfer with MyoD. J. Clin, Invest.    98:2209-2217.-   46. Roest P A., A. C. van der Tuljn, H. B. Ginjaar; RC.    Hoeben, F. B. Hoger-Vorst, E. Bakker, J. T. den Dunnen and G. J. van    Ommen (1996) Application of in vitro Myo-differentiation of    non-muscle cells to enhance gene expression and facilitate analysis    of muscle proteins. Neuromuscul. Disord. 6:195-202.-   47. Havenga M. J., A. A. Lemckert, O. J. Ophorst, M. van    Meijer, W. T. Germeraad, J. Grimbergen, M. A. van Den Doel, R.    Vogels, J. van Deutekom and A. A. Janson, et al. (2002) Exploiting    the natural diversity in adenovirus tropism for therapy and    prevention of disease. J. Virol. 76:4612-4620.-   48. Anderson L. V. and K. Davison (1999) Multiplex Western blotting    system for the analysis of muscular dystrophy proteins. Am. J.    Pathol. 154:1017-1022.-   49. Neugebauer K M et al., J. Cell. Biol. 129:899-908 (1995).-   50. Tacke R and J. L. Manley, Proc. Soc. Exp. Biol. Med. 220:59-63    (1999).-   51. Graveley B. R. et al., Curr Biol. 9:R6-7 (1999).-   52. Misteli T et al., Nature 387:523-527 (1997).-   53. Gorman L, Suter D, Emerick V, et al. Stable alteration of pre    mRNA splicing patterns by modified U7 small nuclear RNAs. Proc.    Natl. Acad. Sci. USA 1998, 4929-4934.-   54. Suter D, Tomasini R, Reber U, et al. Double target antisense U7    snRNAs promote efficient skipping of an aberrant exon in three human    beta thalassemic mutations. Hum. Mol. Genet. 1999, 8:2415-2423.

TABLE 1 Overview of the patients, the AONs and the primer sets used inthis study Targeted RT- Primary Nested Patients Mutations exons AONs^(a)primers^(b) PCR sets^(b) PCR sets^(b) DL 515.2 Deletion exon 45-50 Exon51 h51AON1 h53r h4lf-h53r h42f-h52r DL 363.2 Deletion exon 45-54 Exon 44h44AON1 h55r2 h42f4-h55r2 h44f-h55r 50685.1 Deletion exon 48-50 Exon 51h51AON1 h53r h46f-h53r h47f-h52r DL 589.2 Deletion exon 51-55 Exon 50h50AON1 h58r h47f-h58r h49f-h57r 53914.1 Deletion exon 52 Exon 51h51AON1 h55r h49f-h55r h50f-h54r ″ Exon 53 h53AON1 ″ ″ ″ 50423.1 Pointmutation exon 49 Exon 49 h49AON1 h52r h46f-h52r h47f-h51r ^(a)AONsequences were published previously (23). ^(b)Primer sequences availableupon request.

TABLE 2  Characteristics of the AONs used to study thetargeted skipping of 15 different DMD exons^(a) SEQ IDAntisense sequence Length Exon NO: Name (5′-3′) (bp) G/C % U/C % skipTranscript 1 h2AON 1 comuuuugugaauguuuucuuuu 24 29 75 + OF 2 h2AON 2uuguguauuuacccauuuugug 22 36 68 − OF 3 h29AON 1 Uauccucugaaugucgcauc 2045 65 + IF 4 h29AON 2 gguuauccucugaaugucgc 20 50 60 + IF 5 h40AON 1Gagccuuuuuucuucuuug 19 37 79 + IF 6 h40AON 2 Uccuuucgucucugggcuc 19 5879 + IF 7 h41AON 1 Cuccucuuucuucuucugc 19 47 95 + IF 8 h41AON 2Cuucgaaacugagcaaauuu 20 35 50 + IF 9 h42AON 1 cuugugagacaugagug 17 4741 + IF 10 h42AON 2 cagagacuccucuugcuu 18 50 67 + IF 11 h43AON 1ugcugcugucuucuugcu 18 50 78 − OF 12 h43AON 2 Uuguuaacuuuuucccauu 19 2679 + OF 13 h44AON 1 cgccgccauuucucaacag 19 58 63 + OF 14 h44AON 2uuuguauuuagcauguuccc 20 35 70 + OF 15 h45AON 1 gcugaauuauuucuucccc 19 4274 − OF 16 h45AON 5 gcccaaugccauccugg 17 65 58 + OF 17 h46AON 4bcugcuuccuccaacc 15 60 80 + OF 18 h46AON 8b gcuuuucuuuuaguugcugc 20 4075 + OF 19 h47AON 1 ucuugcucuucugggcuu 18 50 78 − IF 20 h47AON 2cuugagcuuauuuucaaguuu 21 29 67 − IF 21 h48AON 1 uuucuccuuguuucuc 16 3894 − IF 22 h48AON 2 ccauaaauuuccaacugauuc 21 33 62 − IF 23 h49AON 1Cuuccacauccgguuguuu 19 47 74 + IF 24 h49AON 2 Guggcugguuuuuccuugu 19 4768 + IF 25 h50AON 1 cucagagcucagaucuu 17 47 59 + OF 26 h50AON 2ggcugcuuugcccuc 15 67 73 − OF 27 h51AON 1 Ucaaggaagauggcauuucu 20 4045 + OF 28 h51AON 2 ccucugugauuuuauaacuugau 23 30 65 + OF 29 h53AON 1cuguugccuccgguucug 18 61 72 + OF 30 h53AON 2 uuggcucuggccuguccu 18 61 72− OF ^(a)Two AONs were tested per exon. Their different lengths and G/Ccontents (%) did not correlate to their effectivity in exon skipping (1,induced skipping, 2, no skipping). The AONs were directed to purine (A/G)-rich sequences as indicated by their (antisense) U/C content (%).Skipping of the target exons resulted in either an in-frame (IF) or anout-of-frame (OF) transcript. b van. Deutekom et al., 2001 [21].

TABLE 3 Primer sets used for the RT-PCR analyses to detect the skippingof the targeted exons^(a) Primary Nested PCR PCR Target exon RT-primerprimer set primer set  2 h4r h1f1-h4r hlf2-h3r  2 h9r h1f1-h9r h1f2-h8r29 h3lr h25f-h3lr h26f-h30r 40 h44r h38f-h44r h39f-h43r 41 h44rh38f-h44r h39f-h43r 42 h44r h38f-h44r h39f-h43r 43 h47r h41f-h47rh42f-h46r 44 h47r h4lf-h47r h42f-h46r 45 h47r h4lf-h47r h42f-h46r 46h48r h44f-h48r h45f-h47r 47 h52r h44f-h52r h46f-h5lr 48 h52r h44f-h52rh46f-h5lr 49 h52r h44f-h52r h46f-h5lr 50 h52r h44f-h52r h46f-h51r 51h53r h47f-h53r h49f-h52r 53 h55r h50f-h55r h5lf-h54r ^(a)Primersequences are available upon request

TABLE 4 Overview and frequency of the DMD-causing mutations in theLeiden DMD (LDMD) Database, theoretically correctable by skipping one ofthe 12 exons successfully targeted in this study Skippable exonTherapeutic for DMD-mutations: % of deletions No. of nonsense Deletions(exons) in LDMD Duplications % of duplications mutations in (exons)Database (exons) in LDMD Database LDMD Database  2 3-7, 3-19, 3-21 2.9 2 9.0 29 5 40 1 41 4 42 0 43 44, 44-47, 44-49, 3.7 43 3.0 44-51 445-43, 14-43, 19-43, 7.8 44 3.0 30-43, 35-43, 36,-43, 40-43, 42-43, 45,45-54 46 21-45, 45, 47-54, 5.6 47-56 49 1 50 51, 51-53, 51-55 5.2 50 3.051 45-50, 47-50, 48-50, 17.5 51 1.5 49-50, 50, 52, 52-63 53 10-52,45-52, 46-52, 7.5 47-52, 48-52, 49-52, 50-52, 52

TABLE 5 Overview of the patients, the AONs and the primer sets used inExample 3 Targeted RT- Primary PCR Nested PCR Patients Mutations exonsAONs primers^(b) primer sets^(b) primer sets^(b) DL 90.3 Nonsensemutation exon 43 Exon43 h43AON2^(a) h48r h4lf-h48r h42f-h47r Exon 44h44AON1^(a) DL470.2 Deletion exon 46-50 Exon 45 h45AON5 h53r h42f-h53rh43-h52r Exon 51 h51AON2^(a) Exon 45 U-linker h53r h42f-h53r h43f-h52rExon 51 AON^(c) ^(a)Separate AON sequences were published previously(Aartsma-Rus, 2002). ^(b)Primer sequences available upon request ^(c)Ulinker AON consists of h45AON5 linked to h51AON2 by ten uracils.

1. An antisense oligonucleotide of 16 to 80 nucleotides in length,wherein said oligonucleotide is complementary to a consecutive part ofbetween 16 and 50 nucleotides of an sequence internal to exon 55 of thehuman dystrophin pre-mRNA and said oligonucleotide is capable ofinducing skipping of exon 55 in a muscle cell, said oligonucleotidecomprising a modification.
 2. The antisense oligonucleotide of claim 1,wherein said oligonucleotide is 16 to 50 nucleotides in length.
 3. Theoligonucleotide of claim 3, wherein said oligonucleotide is 16 to 24nucleotides in length.
 4. The oligonucleotide of claim 1, wherein theoligonucleotide comprises a 2′-O-methyl-phosphorothioate oligonucleotidemodification.
 5. The oligonucleotide of claim 1, wherein theoligonucleotide consists of a 2′-O-methyl oligonucleotide or a2′-O-methyl-phosphorothioate oligonucleotide.
 6. The oligonucleotide ofclaim 1, wherein the oligonucleotide comprises a morpholine ring.
 7. Theoligonucleotide of claim 6, wherein the oligonucleotide furthercomprises a phosphorodiamidate linkage.
 8. The oligonucleotide of claim1, wherein the oligonucleotide comprises a peptide nucleic acid and/orlocked nucleic acid.
 9. The oligonucleotide of claim 2, wherein saidoligonucleotide is fully complementary to said consecutive part.
 10. Theoligonucleotide of claim 9, wherein the oligonucleotide comprises a2′-O-methyl-phosphorothioate oligonucleotide modification.
 11. Theoligonucleotide of claim 9, wherein the oligonucleotide consists of a2′-O-methyl oligonucleotide or a 2′-O-methyl-phosphorothioateoligonucleotide.
 12. The oligonucleotide of claim 9, wherein theoligonucleotide comprises a morpholine ring and a phosphorodiamidatelinkage.
 13. The oligonucleotide of claim 9, wherein the oligonucleotidecomprises a peptide nucleic acid and/or locked nucleic acid.
 14. Theoligonucleotide of claim 1, wherein said oligonucleotide is capable ofinducing skipping of said exon in primary human myotubes upontransfection of at least 100 nM of said oligonucleotide to said myotubesand incubation of transfected myotubes for at least 16 hours.
 15. Theoligonucleotide of claim 14, wherein skipping is detectable by RT-PCRand nucleic acid sequencing.
 16. An expression vector encoding anoligonucleotide according to claim
 1. 17. A gene delivery vehiclecomprising the expression vector of claim
 16. 18. The oligonucleotide ofclaim 1, wherein said consecutive part comprises a sequence whichassumes both a double-stranded stretch and a single-stranded stretch ofnucleotides, said double-stranded stretch comprising two single-strandednucleic acid stretches of exon 55 that form a duplex.
 19. A method forinducing skipping of exon 55 of the human dystrophin pre-mRNA in amuscle cell in vitro or in vivo, the method comprising contacting saidcell or administering to a human subject comprising said cell anoligonucleotide according to claim 1 for a time and under conditionswhich permit induction of exon 55 skipping.